|Ahead of print publication
Immunohistochemical expression of Ki-67 and Glypican-3 to distinguish aggressive from nonaggressive benign odontogenic tumors
TP Chaturvedi, Kanupriya Gupta, Rahul Agrawal, PG Naveen Kumar, Jatin Gupta
Faculty of Dental Sciences, IMS, BHU, Varanasi, Uttar Pradesh, India
|Date of Submission||05-Apr-2020|
|Date of Decision||30-May-2020|
|Date of Acceptance||14-Jul-2020|
|Date of Web Publication||03-Aug-2021|
Faculty of Dental Sciences, IMS, BHU, Varanasi - 221 005, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: The benign neoplasms are normally slow growing, indolent with no invasive potential. However, there exist a few locally aggressive benign odontogenic tumors that have a tendency to invade and deform the surrounding structures. The exact reason for the aggressiveness of these benign neoplasms remained an enigma. Their biology and clinical expression can often be destructive and ominous. An appropriate treatment protocol needs to be followed to combat the high recurrence rate and aggressiveness of these entities. Aggressive and noniaggressive epithelial odontogenic tumors were analyzed immunohistochemically with Ki-67 and glypican 3 (GPC3).
Materials and Methods: Fifty-nine cases of tumors were divided into aggressive odontogenic tumors (20 solid ameloblastomas, four unicystic ameloblastoma, and 28 keratocystic odontogenic tumors) and nonaggressive odontogenic tumors (five adenomatoid odontogenic tumors and two calcifying cystic odontogenic itumors).
Results: Statistical analysis using Pearson correlation showed Ki-67 to be a better marker for differentiating aggressive from nonaggressive odontogenic tumor as compared to GPC3 (P < 0.001, highly significant), whereas among aggressive tumors, GPC3 turned out to be more useful as compared to Ki-67 (P < 0.001, highly significant).
Conclusion: The present study provides an insight into the different biological behavior of odontogenic tumors, which can thus be helpful in determining the therapy strategies for more aggressive odontogenic tumors.
Keywords: Aggressive, glypican, odontogenic tumors, proliferation
|How to cite this URL:|
Chaturvedi T P, Gupta K, Agrawal R, Naveen Kumar P G, Gupta J. Immunohistochemical expression of Ki-67 and Glypican-3 to distinguish aggressive from nonaggressive benign odontogenic tumors. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=322902
| > Introduction|| |
Odontogenic tumors are the derivatives of epithelial, ectomesenchymal, and/or mesenchymal elements of the tooth-forming apparatus. These include a spectrum of a heterogeneous group of lesions ranging from tumor-like malformations to benign neoplasms and their malignant counterparts, some with metastatic potential. Although they are broadly classified into benign and malignant types, there are odontogenic tumors that are described as benign lesions but display locally aggressive behavior. The word aggressive is often associated with the malignant neoplasms which have the ability to invade the adjacent tissue, thus subsequently resulting in metastasis and finally death if left untreated.
On the other hand, the benign neoplasms exhibit a very characteristic slow, progressive, and self-limiting growth. They are noninvasive, histologically benign with few mitotic cells and high differentiation of cells. The locally aggressive benign tumors are characterized by their inherent potential of local tissue destruction and deformation with severe morbid results.
Ameloblastomas and keratocystic odontogenic tumors (KOTs) are benign tumors derived from the odontogenic epithelium that contain fibrous stroma without odontogenic ectomesenchyme. These tumors are characterized by their locally aggressive behavior and capacity for recurrence. In contrast, adenomatoid odontogenic tumors and calcifying cystic odontogenic tumors are noninvasive tumors characterized by low recurrence rates. Although these tumors are derived from the odontogenic epithelium, they differ in terms of their histogenesis.,
Assessment of proliferation has become popular in histopathology as a means of predicting the behavior of tumors, that is, their likelihood of local recurrence, their metastatic potential, growth of metastases, and thereby the disease-free survival. Immunohistochemical assessment of cell proliferation has advantages over the other techniques, such as tritiated thymidine incorporation and flow cytometry, because the tissue architecture remains intact and proliferating cells can be visualized in relation to other histologic characteristics.
Glypicans (GPCs) are a family of heparin sulfate proteoglycans which are bound to the cell membrane via a glycosylphosphatidylinositol anchor. The GPC gene family consists of six members in mammals. GPC3 is involved in the regulation of cell proliferation and morphogenesis. Although it is abundant in embryonic tissue, GPC3 is limited found in most adult tissues. GPC3 deletion or mutation can disturb the balance between cell apoptosis and proliferation which may result in tumorigenesis. GPC3 upregulation and overexpression have been observed in hepatocellular carcinoma, malignant melanoma, neuroblastoma, and colon cancer.
Furthermore, GPC3 acts as a negative regulator of Hh activity by competing with PTCH1 for SHH binding, and components of the SHH pathway have been investigated in many odontogenic tumors.
However, to our knowledge, there are no studies investigating Ki-67 and GPC3 together in odontogenic tumors. Therefore, the present study was carried out with the objective to determine the immunohistochemical expression of these markers in aggressive and nonaggressive odontogenic tumors.
| > Materials and Methods|| |
Tissues and samples
The present study was carried out comprising a total of 59 cases of tumors which were divided into aggressive odontogenic tumors:
- 20 solid ameloblastoma (8 follicular ameloblastoma, 8 plexiform ameloblastoma, and 4 acanthomatous ameloblastoma),
- 4 unicystic ameloblastoma,
- 28 keratocystic odonttogenic tumor.
Nonaggressive odontogenic tumors:
- 5 adenomatoid odontogenic tumors and
- 2 calcifying cystic odontogenic tumors.
Formalin-fixed paraffin-embedded tissue blocks were retrieved from the archives of Unit of Oral Pathology and Microbiology, Faculty of Dental Sciences, IMS, BHU, Varanasi.
Relevant clinical details were recorded as per the case history pro forma and tabulated. The selected 59 cases were then subjected to hematoxylin and eosin staining for confirmation and then screened immunohistochemically for Ki6-7 and GPC3 positivity.
Immunohistochemical staining was performed using the following antibodies: Ki 67 (MIB 1 clone), GPC 3 (1G12 clone) from immunoglobulin fractions, prediluted, ready to use,monoclonal, Dako, USA. Sections of tissue blocks were cut at 3-μ thickness using Microm Automated Microtome (HM 350 S) and were placed on poly-L-lysine-coated slides for immunostaining. The slides were deparaffinized with three changes of xylene 5 min each, and the endogenous peroxidase activity of the tissue was blocked by placing the tissue in 3% hydrogen peroxide for 30 min. Heat-induced epitope retrieval was done using a microwave oven. The tissue was then incubated with the primary antibody for 60 min. Biotinylated secondary antibody and streptavidin–peroxidase conjugate were added sequentially with the specimen being incubated for 30 min in each of them. Finally, the chromogen diluted in the substrate buffer was added and counterstained with Harris hematoxylin. Staining is seen as a dark brown to black nuclear signal in cells expressing the Ki-67 antigen. Cells exhibiting brown staining in a membrane and/or cytoplasmic location were defined as positive for GPC3 [Figure 1] and [Figure 2]. Representative fields were randomly selected in each stained section. Counting was done under an Olympus compound microscope fitted with an eyepiece ×10 magnification and objective ×40 magnification.
|Figure 1: Ki-67 expression in aggressive odontogenic tumors (a) Keratocystic odontogenic tumor, (b) Unicystic ameloblastoma, (c) Follicular Ameloblastoma, (d) Acanthomatous Ameloblastoma, and (e) Plexiform Ameloblastoma (original magnification ×400)|
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|Figure 2: Glypican 3 expression in aggressive odontogenic tumors (a) Keratocystic odontogenic tumor, (b) Unicystic ameloblastoma, (c) Follicular Ameloblastoma, (d) Acanthomatous Ameloblastoma, and (e) Plexiform Ameloblastoma (original magnification ×400)|
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The intensity was graded based on the number of positive cells seen:
The following scores were attributed:
- Score 0, absence of staining
- Score 1, weak staining
- Score 2, moderate staining
- Score 3, strong staining.
The proportion of staining was defined as the percentage of stained cells (0, 0%; 1, 1% to 25%; 2, 25% to 75%; 3, >75%). Multiplication of the intensity of immunostaining (0–3) by the proportion of stained cells (0 >75%) resulted in the following score:
- <25% of the cells were stained or the staining intensity was weak, the maximum product of the two scores was 3, and immunostaining was classified as low
- At least 25% of the cells were stained or the staining intensity was moderate or strong, the maximum product of the two scores was at least 4, and immunostaining was classified as high (adapted from Dultra et al.).
The data were analyzed using the IBM® SPSS® Statistics 21. The Karl Pearson's correlation coefficient was used to evaluate possible associations of Ki-67 and GPC3 expression between aggressive and nonaggressive odontogenic tumors. A level of significance of 5% was adopted.
| > Results|| |
On statistically analyzing, intensity of Ki-67 stained positive cells in nonaggressive and aggressive odontogenic tumors, Pearson correlation r was 1.000, P < 0.001 (highly significant), indicating a perfect positive correlation between the intensity of Ki-67 positive cells and aggressiveness of the odontogenic tumors.
Intensity of GPC3 stained positive cells in nonaggressive and aggressive odontogenic tumors, Pearson correlation r was 0.700, P < 0.001 (highly significant), indicating a perfect positive correlation between the intensity of GPC3 positive cells and aggressiveness of the odontogenic tumors. The analysis showed the intensity of Ki-67 to be a better indicator of aggressiveness as compared to GPC3 [Graph 1] and [Graph 2].
When Ki-67 and GPC3 were compared among aggressive odontogenic tumors, the intensity of Ki-67 could not be analyzed as all the cases showed the same intensity, whereas the intensity of GPC3 was positively correlated with aggressiveness of odontogenic tumor (r = 0.647, P < 0.001, highly significant), inferring that GPC3 is more sensitive in determining the aggressiveness of odontogenic tumors [Graph 3].
| > Discussion|| |
Locally aggressive benign odontogenic tumors, though benign, possess an inherent tendency to invade and deform. With a higher rate of their recurrence and aggressiveness as compared to other benign tumors, it is crucial to decide upon an effective treatment modality. Furthermore, as these lesions exhibit a higher proliferation rate and invasiveness, early diagnosis and intervention is vital to avoid morbidity and mortality. In the present study, despite the observation of a statistically significant difference between ameloblastomas and KOTs, these aggressive odontogenic tumors exhibited an immunohistochemical distinction of Ki-67 and GPC3 compared to adenomatoid odontogenic tumors and calcifying odontogenic tumors. Thus, these immunohistochemical markers seem to have contributed to invasiveness of those tumors.
The expression of Ki-67 in aggressive odontogenic tumors was in the order, minimum with unicystic ameloblastoma, followed by follicular ameloblastoma, acanthomatous ameloblastoma, and odontogenic keratocyst, and was maximum with plexiform ameloblastoma, when subjected to statistical analysis, resulted to be highly significant.
The present study showed the absence of GPC3 staining in unicystic ameloblastomas. However, despite this difference compared to solid ameloblastomas, these results may not be consistent considering the small number of unicystic cases, although unicystic tumors are less aggressive. Furthermore, plexiform ameloblastoma showed the presence of maximum number of positive cells, followed by follicular and acanthomatous ameloblastoma, thereby affirming to the aggressiveness of plexiform ameloblastoma as compared to other aggressive odontogenic tumors.
The results of our study are consistent with Mendes et al., the authors concluded the role of GPC3 in odontogenic tumors in distinguishing aggressive from nonaggressive odontogenic tumors. Expression of SHH signaling molecules, SHH, PTC, smoothened, and GLI1, has been detected in several odontogenic tumors, suggesting that the SHH signaling pathway plays a role in epithelial–mesenchymal interactions and cell proliferation during the growth of odontogenic tumors as well as during tooth development. GPC3 acts as a negative regulator of Hh activity by competing with PTCH1 for SHH binding. A better understanding of underlying molecular mechanisms will help to predict the course of odontogenic tumors and lead to the development of new therapeutic applications, such as molecular-targeted treatment and patient-tailored therapy, for odontogenic tumors.
Other authors also tried to establish differences between benign odontogenic tumors by showing that calretinin was expressed differently in ameloblastomas and other odontogenic tumors such as adenomatoid odontogenic tumor, ameloblastic fibroma, and odontogenic myxoma. Calretinin has been suggested to be important for the differential diagnosis between solid and unicystic ameloblastomas. Differential expression of calretinin in unicystic ameloblastomas compared to residual cysts, dentigerous cysts, and odontogenic keratocysts has been reported. According to Koneru et al., calretinin is expressed in ameloblastomas and keratocysts but not in adenomatoid odontogenic tumors.
Bologna-Molina et al. demonstrated the presence of another member of the GPC family, GPC-1, in different types of ameloblastomas but found no differences between solid and unicystic cases. In the present study, predominantly cytoplasmic staining was observed in ameloblastomas and KOTs. Ameloblastomas, the different morphological components of the tumors, were stained, similar to the finding of Bologna-Molina R et al. for GPC-1.In contrast, in KOTs, immunostaining was observed in the suprabasal and intermediate layers, which seem to correspond to the proliferative compartment of these tumors as reported in the literature.
This study provided insights into the differential expression of Ki-67 and GPC3 in odontogenic tumors, thereby determining the treatment strategies for aggressive odontogenic tumors. Studies involving a larger number of cases should contribute to the understanding of the role of GPC3 in other odontogenic tumors. Other aspects including recurrence and size of KOTs and ameloblastomas could make clear differences between aggressive and nonaggressive odontogenic tumors as well.
It is important to keep in mind that due to the rarity of histopathologic variants and the low number of cases studied in the present series, the results may be interpreted only as a trend, and it is advisable to study larger series of these variants of ameloblastomas to confirm these observations. Furthermore, further studies suggesting the role of GPC3 in recurrence of OTs and their chances of malignant transformation to ameloblastic carcinoma should be uptaken. Another limitation of this study was that we only have access to the use of the technique of immunohistochemistry and then the limiting factor of the semiquantitative quantification. This work only describes the presence of “in situ” GPC3 protein immunoreactivity and offers possible hypotheses about the functions of GPC3 in OTs. Future long-term studies with more cases, radiographic analysis, and second experimental approaches like molecular techniques determining the rate of recurrence are needed to clarify each possible function of GPC3 that was suggested in this work.
| > Conclusion|| |
The proliferation potential of the epithelium and the overexpression of GPC3 in odontogenic epithelial tumors are quite significant for their clinical behavior. High expressions of GPC3 and Ki-67 accord with their aggressive clinical behavior and a high recurrence rate.
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Conflicts of interest
There are no conflicts of interest.
| > References|| |
Carlson ER,August M,Ruggiero S. Locally Aggressive Benign Processes of the Oral and Maxillofacial Region.Selected Readings in Oral and Maxillofacial Surgery 2004;12:1-52.
Torres-Domingo S, Bagan JV, Jiménez Y, Poveda R, Murillo J, Díaz JM, et al
. Benign tumors of the oral mucosa: A study of 300 patients. Med Oral Patol Oral Cir Bucal 2008;13:E161-6.
Barnes L, Eveson JW, Reichart P, Sidransky D, editors. World Health Organization Classification of Tumors. Pathology and Genetics of Head and Neck Tumors. Lyon: IARC Press; 2005. p. 306-7.
Mello LA, Figueiredo AL, Ramos EA, Gurgel CA, Martins MD, de Figueiredo CR, et al
. CD1a-positive Langerhans cells and their relationship with E-cadherin in ameloblastomas and keratocystic odontogenic tumors. J Oral Pathol Med 2013;42:454-61.
Lee SK, Kim YS. Current concepts and occurrence of epithelial odontogenic tumors: II. Calcifying epithelial odontogenic tumor versus ghost cell odontogenic tumors derived from calcifying odontogenic cyst. Korean J Pathol 2014;48:175-87.
Lee SK, Kim YS. Current concepts and occurrence of epithelial odontogenic tumors: I. Ameloblastoma and adenomatoid odontogenic tumor. Korean J Pathol 2013;47:191-202.
Baserga R. The relationship of the cell cycle to tumor growth and control of cell division: A review. Cancer Res 1965;25:581-95.
Tsai ST, Jin YT. Proliferating cell nuclear antigen (PCNA) expression in oral squamous cell carcinomas. J Oral Pathol Med 1995;24:313-5.
Filmus J, Selleck SB. Glypicans: Proteoglycans with a surprise. J Clin Invest 2001;108:497-501.
Andisheh-Tadbir A, Ashraf MJ, Gudarzi A, Zare R. Evaluation of Glypican-3 expression in benign and malignant salivary gland tumors. J Oral Biol Craniofac Res 2019;9:63-6.
Liu H, Yang C, Lu W, Zeng Y. Prognostic significance of glypican-3 expression in hepatocellular carcinoma: A meta-analysis. Medicine (Baltimore) 2018;97:e9702.
Kumamoto H, Ohki K, Ooya K. Expression of Sonic hedgehog (SHH) signaling molecules in ameloblastomas. J Oral Pathol Med 2004;33:185-90.
Dultra FK, Barros AC, Schaer-Barbosa H, Figueiredo AL, Gurgel CA, Ramos EA, et al
. Immunohistochemical assessment of CD1a-positive Langerhans cells and their relationship with E-cadherin in minor salivary gland tumors. J Oral Pathol Med 2012;41:47-53.
Mendes RB, Dias RB, Figueiredo AL, Gurgel CA, Santana Filho M, Melo LA, et al
. Glypican-3 distinguishes aggressive from non-aggressive odontogenic tumors: A preliminary study. J Oral Pathol Med 2017;46:297-300.
Kumamoto H. Molecular pathology of odontogenic tumors. J Oral Pathol Med 2006;35:65-74.
Altini M, Coleman H, Doglioni C, Favia G, Maiorano E. Calretinin expression in ameloblastomas. Histopathology 2000;37:27-32.
Coleman H, Altini M, Ali H, Doglioni C, Favia G, Maiorano E. Use of calretinin in the differential diagnosis of unicystic ameloblastomas. Histopathology 2001;38:312-7.
Koneru A, Hallikeri K, Nellithady GS, Krishnapillai R, Prabhu S. Immunohistochemical expression of calretinin in ameloblastoma, adenomatoid odontogenic tumor, and keratocystic odontogenic tumor: A comparative study. Appl Immunohistochem Mol Morphol 2014;22:762-7.
Bologna-Molina R, Mosqueda-Taylor A, Molina-Frechero N. Differential expression of glypican-1 in ameloblastoma variants. Appl Immunohistochem Mol Morphol 2015;23:153-60.
[Figure 1], [Figure 2]